Classical nuclear receptors for steroid hormones have additional functions outside of the nucleus to mediate rapid steroid activation of protein phosphorylation signaling cascades and this has been demonstrated to contribute to a number of cellular responses to sex steroids including proliferation. The overall goal of this research is to define mechanisms and the biological role of progesterone receptor (PR) activation of cell signaling pathways. We propose that signaling pathways activated by PR outside of the nucleus couple with nuclear receptors, or other transcription factors, through a regulatory loop that is critical for tight hormonal control of gene expression. We also hypothesize that signaling pathways activated by extra- nuclear PR are prevalent at specific stages of the cell cycle and important for the highly regulated control of cell proliferation by progesterone. Studies in this proposal will use combined approaches of molecular biology, single cell fluorescence imaging and mammary gland reconstitution in the mouse to address these questions in vitro and in vivo with the following specific aims: Aim # 1 will determine the role and mechanisms of PR activation of extra-nuclear signaling pathways in the regulation of progesterone responsive target genes in cell culture systems. Aim #2 will determine by fluorescence cell imaging techniques the intracellular site(s) of PR interaction with c-Src kinase (a key entry point for intersection of PR with signaling pathways) and whether these interactions and rapid signaling are cell cycle specific. Aim #3 will determine the in vivo role of PR activation of signaling pathways in the mouse mammary gland by genetic manipulation of PR signaling in a tissue transplant/reconstitution system. There is increasing evidence and appreciation that progesterone has a role in early stages of breast cancer development and that an imbalance between rapid extra-nuclear signaling and nuclear transcriptional actions of steroid receptors may contribute to the etiology and progression of hormone-dependent cancers. Information gained from this research will advance our understanding of the role of rapid extra-nuclear steroid receptor signaling in breast cancer and will be important for development of pathway selective agents as potential therapeutics. PUBLIC HEALTH RELEVANCE: In addition to their classical role as nuclear transcription factors, steroid receptors have functions outside the nucleus to activate rapid cell signaling pathways. An imbalance between extra-nuclear rapid signaling and nuclear transcription activities of steroid receptors has been implicated to contribute to development and progression of hormone-dependent cancer. The research in this application will provide insights into the mechanisms and role of PR activation of signaling pathways with the long-term goal of developing pathway selective agents as potential therapeutics.